Skip to main content

Viva Voce #2

Hi,
As I said in my previous post Viva Voce #1, my university practical exams are going on, out of the 4 practicals, now I completed 3 :) Today was a day of E.coli, as I got E.coli with all experiments :)
Microbio is my favorite favorite subject, and the lab, I love to be in microbio lab with my microbial buddies :) :P Here we go with my microbiology practical exam :)

I prepared well for my lab exam yesterday night. I made myself clear with all the concepts behind lab techniques and experiments. Had good confidence :) as this is my favorite lab :)
I was given with a question paper saying:

Major experiment: 
Inoculate 10% of E.coli culture in nutrient broth of ph5 and check the growth rate by measuring turbidity and plot a graph.


Minor experiment: 
Perform gram staining of the given culture, observe shape, color and interpret the results.

:) :) :)))) I got a biggggggggg smile when I saw this easy question paper and started with my experiment :)
I cleaned and sterilized the laminar air hood, took my pH5 nutrient broth, cuvettes, pipette. For accounting the turbidity spectrophotometer is used generally by us. So, I took blank as the broth with out any inoculation in a cuvette and I inoculated the broth with given E.coli culture.

After inoculation, I took that inoculated broth in a cuvette for taking OD at 0 minute. I kept the broth flask in shaker at 37 degree celsius. Oh, gosh... :( I forgot to take two blanks :( I realised this only when I started towards the spec :(  What to do.. I was blinking for a minute, then I checked the cupboard for sterile nutrient broth of pH5, to my goodness I found one more flask of sterile pH5 nutrient broth. (Again I got a biggg smile...)

I took another blank from that sterile broth :) and took O.D. I took O.D of the inoculated broth for every 30 minutes, say at 30,60,90,120,150 minutes.

E.coli normally grows at optimum pH of 7. So, in my pH 5 broth, I expected that the growth would decrease with time. But, to my surprise it increased. Growth was increasing with time. I was confused :(
Then, side by side, in the gap of 30 minutes in taking O.D, I completed gram staining too. I was given with E.coli for Gram staining too. Got, beautiful pink rods :) interpreted that E.coli is a gram negative, hence it stains pink :)  ( What a great experiment :P :P )

In between the external examiner called, "Register number 05..."
oo.. I'm that 05.. I rushed to him with my answer sheets. He got my sheets and started probing it.. He started shooting questions then, Here we go with my Viva voce #2

External examiner: Your getting increased growth with time, do you think, optimum pH of E.coli is 5?
Me: No sir, optimum pH of E.coli is 7.


External examiner: Then, your getting increased growth? Justify this? 
Me: (Thought for a while) Sir, it might be due to contamination with some other species which grows at pH5.
( He was convinced with this answer)
E.coli - Gram staining

External examiner: List some preservation techniques?
Me: Stab culture, Glycerol stock, Lyophilisation.

External examiner: What's the purpose of Safranin in Gram staining?
Me: It is a counter stain. Gram positive retains crystal violet while decorising with ethanol, while Gram negative loses it. So, to give a contrast , Safranin is used. Gram positives won't take up safranin.

External examiner: So.. Crystal violet is a basic dye or acidic?
Me: It's a basic dye, sir.

External examiner: Why a basic dye for staining bacteria? why not an acidic?
Me: Sir, normally bacteria have negative charge, so, using an acidic dye which is also negative charged, there will be repulsion and there won't be staining.

External examiner: okay, that's it. you may go.

He was satisfied with my answers :)  An easy Viva voce once again :)

Then I continued with my experiment, got increase of growth, drawn graph and gave inference as " The increase in growth might be due to contamination of the culture with other species which grows at pH5"

And we were given with some 4 questions:
Here are my 4 questions:

1) Interpret the spotter 1. ( Mam gave an EMB - Eosin methylene blue agar plate) 
My interpretation:
The given plate is an EMB - Eosin methylene blue agar plate - divided into two halves.
In one half, there was growth showing metallic zinc color - indicating that it was E.coli culture.
In another half no growth was obtained, indicating that half was inoculated with a gram positive bacteria.
Principle: EMB agar is a selective media, it allows only gram negatives to grow and E.coli gives metallic zinc with EMB agar.

2) How will you prepare 1ml of 1:5 dilution culture? 
Ans: 0.2 ml of culture in 0.8 ml of broth

3) You are isolating 5 different S.aureus cultures, whether it is necessary to check susceptibility for all the five? Why?
Ans: It is necessary to check susceptibility for all the 5, because one may have resistance and other may not have (  when Isolated from different sources)

4) A simple calculation for calculating phenol coefficient was given. I did it right. I don't remember the numbers given, so, I share just the formula: 

Phenol coefficient = (Greatest dilution of disinfectant that kills microbe in 10 mins but not 5 mins) /
(Greatest dilution of phenol that kills microbe in 10 mins but not 5 mins)

Finished successfully and submitted my paper, came out with satisfaction :) :) :)
Just one more practical exam tomorrow, just it is Communication skills practicals! :) :) Yaaaiii.. yaaii.. did well in all the practicals :) Have a good time :) :)) 

Comments

Popular posts from this blog

Lowry Assay Principle and procedure

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P) Why Lowry?   Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes). Oth

Sea Butterfly!

Neih hou!  Don't roll you eyes wondering what it is! "Neih hou" is how you say "hello" in Cantonese. Guess what, I am in Hong Kong and therefore the language Cantonese. I came to Hong Kong this summer as an intern before officially joining as a PhD Student. This is my second experience abroad, far away from home, new language, new culture, I expected me to have a "really" bad cultural shock, but, actually I experienced only 50% of what I expected. For a girl who have used the marina beach only for eating "sundal", bikini in beach was a shock (FEEL FREE TO JUDGE ME!). The first time, I went to the big wave beach here, I was the only one who was totally covered, when everyone was enjoying their Beer, I was slowly sipping my lemon juice (THE ODD ONE OUT!). I realized how beautifully different the world outside is! There is no single standard for right or wrong, it varies in different countries, in different regions around the world. And it

Isolation of monocytes from PBMC (Peripheral Blood Mononuclear Cells) - Principle and protocol

Whenever I'm made to realise that I'm not clear enough or good at something, I try to make myself clear with it. It happened today, during my laboratory examination, I was asked to perform monocyte isolation from a given blood sample, but, unfortunately, I was not very clear with the principle behind it.(but, still I managed to complete the experiment as I know the protocol, but, knowing the principle behind each step of the protocol clearly is very important, isn't it?). But, nothing is wrong in it, I made myself clear with it now. That's good, right? So, let me share with you some basic principle and protocol for isolating monocyte from blood sample. For isolating monocytes, initially we must isolate PBMC (Peripheral bood mono nuclear cells) from the blood sample. Here, let us make few terminologies clear before starting with the principle. Peripheral blood sample  - It is the blood sample obtained from acral areas of body (in general, it is the blood collected